Cladobotric Acids: Metabolites from Cultures of Cladobotryum sp., Semisynthetic Analogues and Antibacterial Activity

Three new polyketide-derived natural products, cladobotric acids G–I (1–3), and six known metabolites (4, 5, 8–11) were isolated from fermentation of the fungus Cladobotryum sp. grown on rice. Their structures were elucidated by extensive spectroscopic methods. Two metabolites, cladobotric acid A (4) and pyrenulic acid A (10), were converted to a series of new products (12–20) by semisynthesis. The antibacterial activities of all these compounds were investigated against the Gram-positive pathogen Staphylococcus aureus including methicillin-susceptible (MSSA), methicillin-resistant and vancomycin-intermediate (MRSA/VISA), and heterogeneous vancomycin-intermediate (hVISA) strains. Results of these antibacterial assays revealed structural features of the unsaturated decalins important for biological activity.

W ith the increase of antibiotic-resistant bacteria worldwide and the lack of new antibiotics, 1,2 there is a continuing need for the discovery and development of effective antibacterial agents. The majority of commonly used antibiotics in both the clinic and agriculture either are natural products or are analogues or derivatives inspired by natural product leads. 2 With significant advances in genome mining there are excellent prospects for discovering new compounds with antibiotic activity, potentially with novel modes of action. 3−7 In the course of screening for antibacterial natural products, we turned our attention to the fungal strain Cladobotryum sp. CANU E1042. Cladobotryum fungi are known to be the causal agents of "cobweb disease" in agriculture 8 and have been reported to produce a number of bioactive secondary metabolites, including cyclodepsipeptides, 9 cladobotric acids, 10 tricyclic derivatives, 11 substituted pyridinediones, 12 cladobotrins, 13 furopyridines, 14 and azatricyclic phosphate esters. 15 In 2006, Munro and co-workers reported the isolation of six unsaturated decalin-type natural products named cladobotric acids A−F (4−9) from the fermentation broth of a New Zealand Cladobotryum species. 10 The absolute configuration of cladobotric acid A (4) was determined using X-ray crystallography of the p-bromo ester derivative. The results of feeding studies with [ 13 C]-labeled precursors were in accord with the proposed polyketide origin of the cladobotric acids. More recently two compounds closely related to the cladobotric acids, pyrenulic acids A and B (10 and 11, respectively), were isolated from a spore-derived mycobiont of a crustose Pyrenula sp. lichen collected in Vietnam, which showed cytotoxic effects against HCT116 human colon carcinoma. 16 Herein we report the isolation and structure elucidation of three new cladobotric acids (1−3) from cultures of Cladobotryum sp. CANU E1042, 10 which are now named cladobotric acids G−I, along with six known natural products (4, 5, 8−11). Structural modifications of the major metabolites cladobotric acid A (4) and pyrenulic acid A (10) via either reduction or treatment with acid gave nine new unsaturated decalins. The structure−activity relationships (SAR) within this family were investigated by establishing antibacterial activity against the Gram-positive bacterial pathogen Staphylococcus aureus.

■ RESULTS AND DISCUSSION
Isolation and Structure Elucidation. Cladobotryum sp. was grown on rice. After 14 days the growth medium was extracted with EtOAc. Purification of the metabolites by successive chromatographic procedures (silica gel, Sephadex LH-20, RP-18, and HPLC) yielded the six known polyketidederived natural products (4, 5, 8−11) as well as three new related compounds, 1−3.
Compound 1 was obtained as a pale yellow solid (4.5 mg) with the molecular formula C 26 H 36 O 5 as determined from the sodium adduct ion [M + Na] + peak at m/z 451.2470 (calcd for C 26 H 36 O 5 Na, 451.2460) in the HRMS spectrum. Its UV spectrum showed an intense absorption band, λ max (log ε), at 302 nm. Its IR spectrum revealed the presence of hydroxy and carbonyl groups (3407 and 1694 cm −1 , respectively). The structure was deduced by detailed analysis of the 1D and 2D NMR data ( Table 1). The 1 H NMR spectrum of 1 displayed signals for seven olefinic protons, including three E-double bonds, six methine protons including two oxygenated methines, three pairs of methylene protons, and five methyl groups. The 13 C NMR data (Table 1) revealed 26 carbon atoms, including four double bonds, of which one was trisubstituted with a signal for the quaternary carbon at δ C 134.0 (C-12), three oxygenated quaternary sp 3 carbons, δ C 73.1 (C-17), 64.5 (C-18), and 62.4 (C-16), and a signal at δ C 169.7 (C-1) assigned to a carboxylic acid. The full assignment was achieved using 2D (COSY, HSQC, and HMBC) NMR experiments, which revealed the partial structures of 1 as a highly substituted, unsaturated decalin with a trienoic acid side-chain at C-8, similar to that of cladobotric acid A (4). 10 The major differences in the 13 C NMR spectra of the two metabolites were the signals at δ C 134.2 (C-15) and 124.3 (C-16) assigned to the 15,16-alkene in cladobotric acid A (4) versus those at δ C 64.  (Table 1 and Supporting Information Figure S11). All previously reported cladobotric acids have a negative optical rotation, and therefore the absolute configuration of 1 was assigned on the basis of its similar negative value ([α] D −70.2 (c 0.1, CHCl 3 )). This new metabolite is now named cladobotric acid G (1).
Compound 2 was obtained as a pale yellow solid (6.5 mg) with a molecular formula of C 27 H 36 O 5 (HRMS m/z 463.2468 [M + Na] + , calcd for C 27 H 36 O 5 Na, 463.2460). The 1 H and 13 C NMR data of 2 (Table 1) closely resembled those of cladobotric acid C (6) 10 except for the primary alcohol at C-12 (δ H 4.15, δ C 76.2) being replaced by a methyl ester (CO 2 CH 3 δ H 3.73, δ C 51. 8, 167.8). Consistent with this, the signal assigned to 11-H which appeared at δ H 5.66 (br s) in 6 was now downfield at δ H 6.94 (br s) in the new metabolite. These assignments were confirmed from HMBC correlations between 11-H/C-9, C-10, C-13, and C-26, between 13-H/C-11, C-12, C-14, and C-26, and between OCH 3 of the methyl ester and C-26. Thus, compound 2 is assembled on the trans decalin system with the C-26 methyl ester and is now named cladobotric acid H.
Compound 3 was obtained as a pale yellow solid (4.5 mg) with a molecular formula of C 26 H 36 O 3 (HRMS m/z 419.2544 [M + Na] + , calcd for C 26 H 36 O 3 Na, 419.2557). The UV, IR, and NMR spectroscopic data were again in agreement with a cladobotric acid metabolite, with data similar to those reported for pyrenulic acid B (11). 16 The only difference between the two structures was the presence of the hydroxylated C-17 in 3 (δ C 79.9) versus the 17-CH (δ C 53.4) in pyrenulic acid B (11). 16 Further characteristic NMR signals included an olefinic proton at δ H 5.16 (19-H, br d, J 9.5 Hz) and two sp 2 carbons at δ C 133.6 (C-18) and 135.0 (C-19) in accord with a trisubstituted 18,19-alkene rather than the 18,19-epoxide characteristic of cladobotric acids A−H (Table 1). HMBC correlations from 19-H to C-18, C-20, and C-23 and from 24-H 3 to C-17, C-18, and C-19 confirmed the presence of the 18,19-alkene in 3. The E geometry was confirmed by NOE correlations between 20-H/24-H 3 . The absolute configuration was assigned on the basis of the negative value of the optical rotation [α] D −60.4 (c 0.15, CHCl 3 ), and compound 3 is thus named cladobotric acid I.
New Cladobotric Acid Analogues (12−20) Produced by Semisynthesis. The major metabolites isolated from extracts of Cladobotryum sp. grown on rice were cladobotric acid A (4) (600 mg) and pyrenulic acid A (10) (85 mg), providing sufficient material to use as starting materials for the semisynthesis of analogues for structure−activity studies on this family of polyketide-derived natural products. Reduction of 4 with H 2 and 10% Pd on C gave a complex mixture of products, from which two pure compounds, 12 and 13 (4.5% and 13.5% yield, respectively), were isolated using reversephase HPLC (Scheme 1). In both cases the 8-trienoic acid side-chain of cladobotric acid A had been reduced, giving 12 as one of the products. In the second compound (13) it was evident that one of the trisubstituted alkenes had also been reduced, giving a single diastereomer. Extensive 2D NMR investigations revealed that the 11,12-alkene had been reduced, giving the equatorial methyl group at C-12, as determined from the coupling constants for 13-H ax (app. q, J 12.5 Hz) that were in accord with a geminal and two axial−axial couplings and from NOE correlations between 8-H/14-H and 12-H/14-H.
Next, reduction of the carboxylic acid of 4 was investigated via generation of the mixed anhydride using propionyl chloride in the presence of i Pr 2 EtN followed by treatment with NaBH 4 in MeOH (Scheme 1). Two products were obtained, which were purified by HPLC to give primary alcohol 14 and methyl ester 15 (from reaction of the mixed anhydride with MeOH) in 11% and 48% yield, respectively.
Attention was then turned to the reaction of cladobotric acid A (4) with HCl in MeOH/H 2 O. Three products were isolated by reverse-phase HPLC, and their structures confirmed by extensive spectroscopic studies (Scheme 2). Compound 16 Pyrenulic acid A (10) was treated under the same acidic conditions as cladobotric acid A (4), and the products were isolated by HPLC (Scheme 2). In this case two products, 19 and 20 (27% and 15% yield, respectively), were fully characterized, with both possessing a C-17 side-chain incorporating an allylic alcohol formed by the acid-mediated rearrangement of the 18,19-epoxide, with NOE experiments confirming retention of stereochemistry at C-19 (as also found in ester 18). For compound 19 hydration of the 11,12-alkene had also occurred, giving the tertiary alcohol at C-12.
Bioactivity Screening. With a series of new and known cladobotric acids as well as semisynthetic derivatives available, their antibacterial activities were assessed against the Gram-  Table 2. None of the compounds tested revealed any activity against E. coli (data not shown). In contrast, with the exception of cladobotric acid F (9), all compounds tested showed detectable activity against all three S. aureus strains at concentrations ≤ 128 μg/mL. Overall, significant (i.e., minimum inhibitory concentrations (MIC) values more than one dilution apart) strain-dependent activity differences were apparent only for compounds 11 and 19, where in both cases potency increased for the hVISA, compared to the MSSA strain. Pyrenulic acid B (11) showed significant antibacterial activity against both antibioticsusceptible (MSSA) and -resistant (MRSA/VISA and hVISA) S. aureus strains with MIC values ranging from 4 to 16 μg/mL, while cladobotric acids A (4) and I (3) and pyrenulic acid A (10) exhibited moderate activities (MIC values from 16 to 64 μg/mL) ( Table 2). Analysis of the results of assay data for these compounds suggested that compounds lacking an 18,19-epoxide showed enhanced antibacterial activity, for example, comparing 11, with an 18,19-alkene (MIC values from 4 to 16 μg/mL), with the pyrenulic acid A (10) (MIC values 16 to 32 μg/mL). The presence of a 17-OH leads to a decrease in activity compared to the analogous natural products with a 17-H, as evidenced by the decrease in activity of 4, 8, and 3 (MIC range 16−128 μg/mL) in comparison with 10, 2, and 11, (MIC range 4−64 μg/mL; Table 2). Compounds containing C-26 methyl esters (e.g., cladobotric acids E, F, and H, 8, 9, and 2, respectively) tend to exhibit reduced activity (MICs ≥ 64 μg/mL). A similar pattern of antimicrobial activity was observed against Bacillus subtilis compared with S. aureus (Supporting Information, Table S1).
To further analyze the structure−activity relationship of this class of cladobotric acids, antibacterial activities of the semisynthetic derivatives 12−20 were examined ( Table 2). The most active compound of all those tested was allylic alcohol 20 (MIC value 4 μg/mL), which lacks the 18,19epoxide and has a 17-H rather than 17-OH, in accordance with the SAR results obtained from studies on the natural products. Reduction of the triene in the C-8 side-chain has little effect on bioactivity (comparing the activities of 4 and 12), but the carboxylic acid at C-1 appears important, as methyl esters 9, 15, 16, and 18 all exhibited reduced activity, e.g., comparing 8 and 9 (MICs of 128 and >256 μg/mL, respectively).
In conclusion, the three new cladobotric acids G−I (1−3) in addition to six known metabolites (4, 5, 8−11) have been isolated from Cladobotryum sp. CANU E1042, and their structures confirmed by spectroscopic methods. The major metabolites, cladobotric acid A (4) and pyrenulic acid A (10), were converted to a series of novel analogues by semisynthesis. The antibacterial activities against methicillin-and vancomycin-susceptible and resistant S. aureus bacteria (MSSA, MRSA/ VISA, and hVISA) were tested, indicating that anti-Grampositive activity was largely independent of methicillin and vancomycin susceptibility and revealing key structural features for biological activity. A carboxylic acid at C-1 was important (cf. a methyl ester or alcohol at C-1 significantly reduced activity), and in general compounds lacking a 17-OH tended to be more active. The 18,19-epoxide does not appear to be important for bioactivity. Indeed, compounds lacking this moiety (e.g., 11 and 20) exhibited greater activity. As polyketides with an unusual carbon folding pattern, 10,17 further studies on the biosynthetic gene cluster encoding cladobotric acid biosynthesis are ongoing in our laboratories, with the Journal of Natural Products pubs.acs.org/jnp Article biosynthetic pathway likely to be similar to the those for related compounds such as fusarielin 18 and burnettiene A. 19 ■ EXPERIMENTAL SECTION General Experimental Procedures. Optical rotations were measured using a Bellingham and Stanley Ltd. ADP220 polarimeter. UV spectra were recorded in MeOH on a PerkinElmer Lambda 25 UV/vis spectrometer. IR spectra were obtained using a PerkinElmer Spectrum One FT-IR spectrometer as a film on KBr discs. NMR spectra were recorded on a Bruker Advance III HD Cryo 500 MHz spectrometer with TMS (tetramethylsilane) as the reference. Full assignment of NMR data was achieved using 2D experiments including COSY ( 1 H− 1 H correlation spectroscopy), HSQC (heteronuclear single quantum coherence), HMBC (heteronuclear multiplebond correlation), and NOESY (nuclear Overhauser effect spectroscopy). HRESIMS (high-resolution electrospray ionization mass spectrometry) data were recorded on a MicrO-TOF II (Bruker, Daltonics) mass spectrometer. Silica gel (Merck, 63−200 μm particle size), RP-18 (Merck, 40−63 μm particle size), and Sephadex LH-20 were used for column chromatography. TLC (thin layer chromatography) was carried out with silica gel 60 F 254 and RP-18 F 254 plates. HPLC (high-performance liquid chromatography) was carried out using a Waters system using a Phenomenex Kinetex C 18 column (10 × 250 mm, 5 μm particle size). Detection was achieved by a Waters 2998 diode array, a Waters Quattro Micro ESI mass spectrometer, and a Waters 2424 evaporative light scattering detector. All solvents used for extraction and isolation were of analytical grade.
Fungal Material. The fungal strain Cladobotryum sp. CANU E1042 was isolated from a podocarp forest near Hokotika, New Zealand, and provided to us by Munro and co-workers. 10 Fermentation, Extraction, and Purification. The fungus Cladobotryum sp. CANU E1042 was inoculated in a 500 mL Erlenmeyer flask containing 100 mL of a PDB medium (2.4% potato dextrose broth). The flask was incubated statically at 18°C for 3 days. Aliquots of 20 mL of this seed culture were transferred to five Erlenmeyer flasks, each containing white rice (100 g), soaked in H 2 O (100 mL) and autoclaved as a solid production medium. The flasks were incubated statically at 18°C for 14 days. The solid fermentation was then extracted with EtOAc (3 L) by blending and sonicating for 30 min at room temperature. The extract was filtered and concentrated under vacuum to obtain 8.0 g of crude extract. This crude extract was then chromatographed using a silica gel column (4 × 30 cm; 63−200 μm particle size) and eluted with an n-hexane/ acetone series (9:1, 8:2, ..., 1:9, each 0.5 L) to yield seven fractions (F1: 1.5 g; F2: 1.2 g; F3: 0.8 g; F4: 0.6 g; F5: 1.6 g; F6: 0.4 g; F7: 0.5 g). Fractions F4−F6 showed inhibitory effects on a diffusion paper disc assay against S. aureus (Mu50). These fractions were analyzed to characterize which compounds were responsible for this antibacterial activity. Fraction F4 was applied to an RP-18 column (3 × 20 cm; 40 μm particle size) and eluted with a stepwise gradient of MeOH   Conversion of Cladobotric Acid A (4) to Compounds 14 and 15. A solution of 4 (50 mg, 0.125 mmol) in THF (2 mL) was added with EtCOCl (15 mg, 0.162 mmol) and DIPEA (32 mg, 0.25 mmol) and stirred under a N 2 atmosphere at 0°C for 30 min. The reaction mixture was then filtered and concentrated under reduced pressure. The crude reaction mixture was dissolved in MeOH (2 mL), treated with NaBH 4 (24 mg, 0.625 mmol), and stirred at −78°C for 3 h. On completion of the reaction, it was quenched by the addition of a saturated NH 4 Cl solution (2 mL), diluted with H 2 O (8 mL), and extracted with EtOAc (3 × 10 mL). The combined organic extracts were washed with brine (20 mL), dried over anhydrous MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified by HPLC (0−15 min: 82% MeCN, 15−20 min: 82−100% MeCN) to yield compounds 14 (t R = 12.5 min, 5.5 mg, >98% purity) and 15 (t R = 15.8 min, 23.5 mg, >98% purity).